Virus Growing In Hypoxic Cell Or Virus Vector Expressing Gene Therein

Abstract

The present invention provides a virus or a viral vector capable of expressing a gene specifically in a cell having replication ability in a hypoxic state such as a cancer stem cell and injuring the cell, and a pharmaceutical composition comprising the same. Specifically, the present invention provides a virus or a viral vector which comprises a gene encoding a fusion protein of an ODD and a protein essentially required for viral proliferation, and a pharmaceutical composition comprising the same.

Description

TECHNICAL FIELD

[0001] The present invention relates to a virus that comprises a gene encoding a fusion protein of an ODD and a protein essentially required for viral proliferation, a method for producing a virus that uses the virus, a virus or a viral vector that is obtainable by the method, and a pharmaceutical composition comprising the virus or viral vector for treating or preventing a disease characterized by a cell in a hypoxic state. Further, the present application claims the benefit of priority from Japanese Patent Application No. 2008-7179, filed on Jan. 6, 2008, and the entire content of this Japanese Patent Application No. 2008-7179 is incorporated herein by reference.

BACKGROUND ART

[0002] During the course of previous research, the inventors of the present invention modified herpes simplex virus type I (HSV-1) gene to enable the virus to only proliferate in cells in the case of having infected cells that express calponin gene, namely smooth muscle cells growing in leiomyoma, mesothelioma or sites of vascular stenosis, and as a result thereof, developed a method for expressing genes within infected cells and disrupting infected cells (Patent Document 1).

[0003] Cancer cells present in a hypoxic environment within tumors are unresponsive to radiotherapy or chemotherapy, and have the ability to metastasize to distant organs. In addition, cancer stem cells have been reported to be present in cell populations having self-replicating ability in hypoxic regions of bone marrow in acute myelogenous leukemia (Non-Patent Document 1). Moreover, the presence of cancer stem cells has also been recently reported in solid cancers such as breast cancer, brain tumors and colorectal cancer (Non-Patent Document 2). In general, cancer stem cells are considered to be cancer cells that are resistant to therapy, are highly metastatic, and are able to self-replicate even in hypoxic environments (Non-Patent Document 3). Thus, the development of a treatment method is sought that enables cancer cells present in hypoxic regions such as cancer stem cells to be selectively disrupted.

[0004] Research has been conducted on a method for stably expressing arbitrary proteins under hypoxic conditions by using an oxygen-dependent degradation domain (ODD) that serves as a marker for protein degradation at normal oxygen partial pressure present in the amino acid sequence of transcription factor HIFI.alpha., which stabilizes only in cells in a hypoxic state. For example, a method has been developed for inducing apoptosis in cancer cells by producing a fusion gene in which an ODD sequence has been added to the amino terminal of the apoptosis stimulating factor, caspase 3, and activating the caspase 3 only in cells in a hypoxic state, and a method has been developed for expressing diphtheria toxin protein (Non-Patent Documents 4 and 5). However, these methods not only target abnormal cells such as cancer cells, but target normal cells as well. In addition, since these methods involve direct administration of protein whose expression has been inhibited by an ODD sequence, there is the disadvantage of weak therapeutic effects since expression is unable to be sustained due to degradation and the like. Thus, there is a need to develop a method that allows obtaining specific and potent therapeutic effects in target cells. In addition, although adenovirus has previously been produced that is capable of replicating only in cells under hypoxic conditions by coupling E1A gene downstream to the promoter sequence, Hypoxia-Responsive Element (HRE), which responds to HIF1.alpha., there is concern over toxic activity against normal cells due to insufficient on-off control of oxygen partial pressure-dependent promoter activity (Non-Patent Document 6). [0005] Patent Document 1: Japanese Patent Application No. 2006-205006 [0006] Non-Patent Document 1: Sipkins D. A. et al. Nature 435, 969-973, 2005 [0007] Non-Patent Document 2: Ailles L. E. and Weissman I. L. Curr. Opin. Biotech. 18, 460-466, 2007 [0008] Non-Patent Document 3: De Toni F. et al. Oncogene 25, 3113-3122, 2006 [0009] Non-Patent Document 4: Harada H. et al. Cancer Res. 62, 2013-2018, 2002 [0010] Non-Patent Document 5: Koshikawa N. and Takenaga K. Cancer Res. 65, 11622-11630, 2005 [0011] Non-Patent Document 6: Cuevas Y. et al. Cancer Res. 61, 6877-6884, 2003

DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

[0012] An object of the present invention is to provide a virus for specifically injuring cells such as cancer stem cells in a hypoxic state, a viral vector that expresses more genes in cells in a hypoxic state, and preferably in cells having the ability to replicate in a hypoxic state, and a pharmaceutical composition comprising the virus and the viral vector.

Means for Solving the Problems

[0013] As a result of conducting extensive studies with the foregoing in view, the inventors of the present invention fused an oxygen-dependent degradation domain (ODD) with a protein essentially required for viral proliferation, and produced a virus that comprises a gene encoding the fusion protein. Although the fusion protein is degraded following recognition by the ubiquitin-proteasome system as a result of the fusion protein being modified by hydroxylation of a proline residue in the ODD sequence by proline hydroxylase in the case the virus has infected a cell at normal oxygen partial pressure, the fusion protein is not degraded under hypoxic conditions such as at an oxygen partial pressure of 10 mmHg or less. Thus, it was found that the virus does not proliferate in cells at normal oxygen partial pressure, but rather proliferates specifically in the case of having infected cells growing in a hypoxic state, that the gene carried by the virus is expressed, and that specific cytolytic action can be made to occur, thereby leading to completion of the present invention.

[0014] Namely, the present invention provides the inventions indicated in (1) to (16) below:

(1) a virus or viral vector, comprising: a gene encoding a fusion protein of an oxygen-dependent degradation domain (ODD) and a protein essentially required for viral proliferation; (2) the virus or viral vector described in (1), wherein the protein essentially required for viral proliferation is selected from the group consisting of herpes virus ICP4, .gamma.34.5, adenovirus E1A, E1B, retrovirus LTR R and U5; (3) the virus or viral vector described in (2), wherein the protein essentially required for viral proliferation is ICP4; (4) the virus or viral vector described in any of (1) to (3) which has a deficiency in ribonucleotide reductase (RR); (5) a method for producing a virus or viral vector, comprising the steps of: [0015] (a) infecting a cell with the virus described in any of (1) to (4); [0016] (b) culturing the cell in a hypoxic state; and [0017] (c) recovering the virus that has proliferated; (6) a virus or viral vector, which is obtainable by the method described in (5); (7) the virus or viral vector described in (6) which is of the strain d12.ODD.DELTA.RR; (8) a pharmaceutical composition for treating or preventing a disease characterized by a cell having replicating ability in a hypoxic state, which comprises the virus or viral vector described in any of (1) to (4) or the virus or viral vector described in (6) or (7); (9) the pharmaceutical composition described in (8), wherein the disease is selected from the group consisting of cancer, pulmonary fibrosis, pulmonary hypertension, and vascular stenosis in ischemic heart disease or ischemic brain disease; (10) the pharmaceutical composition described in (9), wherein the disease is cancer; (11) the pharmaceutical composition described in (10) which targets a cancer stem cell present in cancer; (12) the pharmaceutical composition described in any of (8) to (11), wherein the hypoxic state is a state in which oxygen partial pressure is 10 mmHg or less; (13) a method for treating or preventing a disease comprising: administering to a subject the virus or viral vector described in any of (1) to (4) or the virus or viral vector described in (6) or (7), wherein the disease is characterized by a cell in the subject having replicating ability in a hypoxic state; (14) a use of the virus or viral vector described in any of (1) to (4) or the virus or viral vector described in (6) or (7) in manufacturing a medicament for treating or preventing a disease in a subject, wherein the disease is characterized by a cell in the subject having replicating ability in a hypoxic state; (15) a fusion protein of an ODD and a protein essentially required for viral proliferation; and, (16) a polynucleotide encoding the protein described in (15).

Effect of the Invention

[0018] Thus, according to the present invention, cells such as cancer stem cells in a hypoxic state, and particularly cells having the ability to replicate in a hypoxic state, can be specifically injured, thereby making it possible to effectively treat or prevent diseases such as cancer, pulmonary fibrosis, pulmonary hypertension or vascular stenosis in ischemic heart disease or ischemic brain disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 indicates expressions of EGFP, HIF1.alpha. and ICP4 after culturing human gastric cancer cell line AZP7a, transfected with a CMV-NLS-ODD-ICP4-IRES-EGFP-polyA DNA fragment, in RPMI medium containing 10% FBS at normal oxygen partial pressure (O.sub.2: 20%) or low oxygen partial pressure (O.sub.2: 1%) as observed with a fluorescence microscope;

[0020] FIG. 2 indicates plaque formation by HSV-1 virus in human gastric cancer cell line AZP7a, co-transfected with DNA of ICP4-deficient HSV-1 mutant strain d120 and a CMV-NLS-ODD-ICP4-IRES-EGFP-polyA DNA fragment, at normal oxygen partial pressure (O.sub.2: 20%) or low oxygen partial pressure (O.sub.2: 1%);

[0021] FIG. 3 indicates construction of d12.ODD.DELTA.RR;

[0022] FIG. 4 represents the proliferation ability of HSV-1 virus as total plaque area when Vero cells are infected with purified d12.ODD.DELTA.RR and cultured for 24 hours at normal oxygen partial pressure (O.sub.2: 20%) or low oxygen partial pressure (O.sub.2: 1%), and indicates that d12.ODD.DELTA.RR proliferated well under conditions of low oxygen partial pressure (O.sub.2: 1%);

[0023] FIG. 5 is a Western blot indicating expression of ICP4 protein when human gastric cancer cell line AZP7a was infected with purified d12.ODD.DELTA.RR at a multiplicity of infection (MOI) of 0.1 and cultured for 48 hours at normal oxygen partial pressure (O.sub.2: 20%) or low oxygen partial pressure (O.sub.2: 1%), and indicates that ICP4 was only expressed at low oxygen partial pressure (O.sub.2: 1%);

[0024] FIG. 6 shows the results of viral replication analysis indicating ganciclovir sensitivity when Vero cells were infected with a purified d12.ODD.DELTA.RR at an MOI of 0.01 and cultured for 26 hours at normal oxygen partial pressure (O.sub.2: 20%) or low oxygen partial pressure (O.sub.2: 1%);

[0025] FIG. 7 indicates immunohistochemical results showing expression of ICP4 protein and the presence of HSV-1 envelope antigen indicating proliferation of d12.ODD.DELTA.RR in a hypoxic region labeled with pimonidazole (injection of d12.ODD.DELTA.RR at 1.times.10.sup.7 pfu) located in a tumor in which human gastric cancer cell line AZP7a was transplanted into the abdominal cavities of SCID mice;

[0026] FIG. 8 indicates antitumor effects of d12.ODD.DELTA.RR against a tumor in which cultured human mesothelioma cell line MSTO was transplanted subcutaneously into the backs of SCID mice (intratumoral injection of d12.ODD.DELTA.RR at 1 to 2.times.10.sup.3 pfu/injection), and tumor volume was determined by quantifying activity of luciferase transfected into the tumor as a photon count by real-time in vivo imaging;

[0027] FIG. 9 indicates antitumor effects of d12.ODD.DELTA.RR against tumors in which primary-cultured human leiomyoma cells established from surgical specimens (n=8) were transplanted subcutaneously into the backs of SCID mice (d12.ODD.DELTA.RR initially injected intratumorally at 1 to 2.times.10.sup.3 pfu/injection, and subsequently injected 17 times at 3 to 4 day intervals) as compared with d12.CALP.DELTA.RR (d12.CALP.DELTA.RR initially injected intratumorally at 1 to 2.times.10.sup.4 pfu/injection, and subsequently injected 17 times at 3 to 4 day intervals), with the left side of the drawing indicating changes in tumor volume, and the right side of the drawing indicating residual tumor cells present in the tumor mass on day 82 following the start of treatment as observed with calponin immunostaining; and

[0028] FIG. 10 shows the results of a viral replication analysis in which cultured GIST cells were divided into fractions positive and negative for the cancer stem cell surface marker, CD133, using AutoMACS Pro (Miltenyi), followed by culturing in Petri dishes and infecting with purified d12.ODD.DELTA.RR at MOI of 0.01 to 0.0001.

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] The present invention in one embodiment relates to a virus or viral vector comprising a gene encoding a fusion protein of an ODD and a protein essentially required for viral proliferation. As was previously described, an ODD (oxygen-dependent degradation domain) refers to a domain that serves as a marker for protein degradation by the ubiquitin-proteasome system at normal oxygen partial pressure present in the amino acid sequence of transcription factor HIF1.alpha., which stabilizes only in a hypoxic state. The amino acid sequence of the ODD is shown in SEQ ID NO: 1, while the nucleic acid sequence thereof is shown in SEQ ID NO: 2. The ODD used in the present invention is composed of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence indicated in SEQ ID NO: 1, and contains a nuclear localization signal (NLS) composed of 23 amino acids and the amino acid proline, which functions as a marker of protein degradation, on the amino terminal thereof. That amino acid sequence is shown in SEQ ID NO: 3. In addition, the nucleic acid sequence encoding the NLS-ODD sequence used in the present invention contains a Kozak consensus sequence on the 5' terminal thereof. That nucleic acid sequence is shown in SEQ ID NO: 4.

[0030] The protein essentially required for viral proliferation includes protein not only essentially required for viral proliferation, but also for replication of viral vector and/or viral nucleic acids. Various proteins are known as such proteins, and examples include, but are not limited to HSV-1 ICP4 and .gamma.34.5, adenovirus E1A and E1B, retrovirus LTR R and U5, and herpes simplex virus ribonucleotide reductase (RR). The amino acid sequences of these proteins are known. The protein essentially required for viral proliferation used in the present invention comprises a protein that is composed of an amino acid sequence in which one or several, such as 9, 8, 7, 6, 5, 4, 3 or 2, amino acids have been deleted, substituted or added in the natural amino acid sequence, and that retains the function of the protein. Since the virus or viral vector of the present invention is used to injure infected cells by being replicated within the infected cells, the virus is preferably a replicative virus. In addition, the virus may be a DNA virus or an RNA virus. The mechanism by which infected cells are injured includes, for example, direct cytolysis due to viral proliferation, induction of apoptosis of virus-infected cells, activation of the immune system and the like.

[0031] The fusion protein of an ODD and a protein essentially required for viral proliferation is a protein in which these two proteins exist as a single polypeptide and are fused so as to retain the functions of each protein. A person with ordinary skill in the art is able to produce such fusion protein based on the sequences of the ODD and the protein essentially required for viral proliferation. Since such fusion protein is able to be degraded incidental to degradation by the ubiquitin-proteasome system of ODD, the presence and/or amount of the fusion protein can be controlled dependent on oxygen partial pressure of infected cells by using the virus or viral vector of the present invention. Preferably, the virus or viral vector of the present invention can be used as a vector that allows the expression of an arbitrary exogenous gene by coupling that gene through an internal ribosomal entry site (IRES) (U.S. Pat. No. 4,937,190) downstream of a gene encoding the sequence of the protein essentially required for proliferation, and allows expression of a greater number of carried genes in cells in a hypoxic state, preferably in cells that have the ability to replicate in a hypoxic state, and for example, cancer stem cells.

[0032] Preferably, the virus or viral vector of the present invention has a deficiency in ribonucleotide reductase (RR). This deficiency refers to a state in which the function of RR has been lost, and is caused by, for example, deletion, insertion or mutation of a nucleotide in a gene encoding RR. RR is an enzyme that functions during the course of DNA synthesis, and is produced in large amounts in actively growing cells such as cancer cells. Thus, a deficiency in RR causes replication of the virus of the present invention to be carried out more frequently in actively growing cells such as cancer cells than in normal cells. In addition, as was previously described, since cells resistant to existing treatment methods, such as cancer stem cells, exist in a hypoxic state and have the ability to self-replicate, the virus or viral vector of the present invention is able to specifically injure growing cells and/or self-replicating cells in a hypoxic state, including cancer stem cells, as a result of having an ODD and being deficient in RR.

[0033] Preferably, the virus or viral vector of the present invention does not contain a gene encoding a protein essentially required for viral proliferation, such as a gene encoding RR, other than a gene encoding the protein essentially required for viral proliferation contained in the fusion protein. Virus replication can be controlled according to oxygen partial pressure more favorably by not containing such a gene.

[0034] In another embodiment, the present invention relates to a method for producing a virus or viral vector that comprises: [0035] (1) infecting a cell with the above-mentioned virus of the present invention; [0036] (2) culturing the cell in a hypoxic state; and, [0037] (3) recovering the virus that has proliferated. Introduction of viral genes of the present invention into cells is carried out by ordinary virus infection. Cells infected by the virus may be any cells provided that the virus is able to replicate within the cells.

[0038] A hypoxic state refers to a state in which oxygen partial pressure is lower than the oxygen partial pressure of the normal proliferation environment of the virus or lower than the normal oxygen partial pressure within a cell, and for example refers to a state in which oxygen partial pressure is about 20 mmHg or less (or about 2% or less as oxygen partial pressure), and preferably a state in which oxygen partial pressure is about 10 mmHg or less (or about 1% or less as oxygen partial pressure). The fusion protein of ODD and the protein essentially required for viral proliferation is stabilized by culturing virus-infected cells in such a hypoxic state, and as a result, viral proliferation is begun, increased or accelerated. Culturing conditions other than a hypoxic state are suitably selected dependent on, for example, the cells infected by the virus.

[0039] The virus that has proliferated is recovered from the culture supernatant according to means or methods known among persons with ordinary skill in the art. The resulting virus can be infected into target cells in a hypoxic state, and preferably target cells that have the ability to replicate in a hypoxic state, and be allowed to proliferate for a fixed period of time enabling it to specifically injure the cells, or the virus or viral vector of the present invention can be obtained in a culture supernatant or in a supernatant obtained by lysing the virus-infected cells and centrifuging.

[0040] In another embodiment, the present invention relates to a virus or viral vector that is obtainable by a method for producing the above-mentioned virus or viral vector of the present invention. The virus or viral vector of the present invention efficiently proliferates in cells in a hypoxic state, and preferably in cells that have the ability to replicate in a hypoxic state. In addition, in the case the virus used in the method for producing the virus or viral vector of the present invention is HSV-1 deficient in RR, the virus or viral vector of the present invention is able to proliferate more favorably in actively growing cells such as cancer cells. Preferably, the virus or viral vector of the present invention is of strain d12.ODD.DELTA.RR. This strain d12.ODD.DELTA.RR will be described in detail herein-below. In addition, the virus or viral vector of the present invention is obtainable by infecting cells with the virus or viral vector of the present invention and causing viral nucleic acids to be replicated within the cells.

[0041] In a further embodiment, the present invention relates to cells that have been infected or introduced with the virus or viral vector of the present invention as previously described. Infection or introduction of the virus or viral vector of the present invention into cells can be carried out using any known methods. Cells that have been infected or introduced with the virus or viral vector of the present invention can be used to induce an immune response to the cells by being injected into the body.

[0042] In a still further embodiment, the present invention relates to a pharmaceutical composition for treating or preventing a disease characterized by cells being in a hypoxic state, and preferably cells having a replicating ability in a hypoxic state, comprising: the above-mentioned virus or viral vector of the present invention. Although previously defined, a hypoxic state preferably refers to a state in which oxygen partial pressure is about 10 mmHg or less (or about 1% or less as oxygen partial pressure). Examples of diseases characterized by cells having the ability to replicate in a hypoxic state include, but are not limited to, all forms of cancer, pulmonary fibrosis, pulmonary hypertension and vascular stenosis in ischemic heart disease or ischemic brain disease. In the case of cancer, for example, cells such as cancer stem cells have the ability to replicate in a hypoxic state. Since only these cells are considered to have the ability to allow cancer to recur or metastasize and be resistant to radiotherapy and chemotherapy, use of the pharmaceutical composition of the present invention enables diseases such as cancer that are resistant to existing treatment methods to be treated, and enables prevention of metastasis thereof.

[0043] The virus or viral vector contained in the pharmaceutical composition of the present invention is characterized by replicating or proliferating in cells in a hypoxic state, and preferably in cells that have the ability to replicate in a hypoxic state. Thus, the pharmaceutical composition comprising the virus or viral vector of the present invention is able to target only cells in a hypoxic state. More specifically, in the case a virus is contained in the pharmaceutical composition of the present invention, the pharmaceutical composition is able to injure only cells in a hypoxic state as a result of the virus proliferating in those cells. In the case a viral vector is contained in the pharmaceutical composition of the present invention, a specific gene can be expressed only in cells in a hypoxic state. Since the viral vector is preferably replicated, the amount of gene expressed in the cells in a hypoxic state is sustained at a high level.

[0044] Moreover, in the case the virus or viral vector contained in the pharmaceutical composition of the present invention has a deficiency in RR, the pharmaceutical composition of the present invention is able to more specifically injure actively growing cancer cells, and preferably cells such as cancer stem cells having the ability to self-replicate.

[0045] The pharmaceutical composition of the present invention may also contain various components such as a carrier, excipient or additive in addition to containing the virus or viral vector of the present invention. The components preferably increase infection efficiency of the virus or viral vector, and are suitably selected depending on various factors such as the disease, state of the target cells to be treated, or the administration mode. The amount and number of administrations of the virus or viral vector contained in the pharmaceutical composition of the present invention are suitably selected depending on the disease, target cells, subject status, administration mode and the like. In addition, known therapeutic agents of a disease to be treated or prevented may also be used in the pharmaceutical composition of the present invention or concomitantly with the pharmaceutical composition of the present invention.

[0046] In a yet further embodiment, the present invention relates to a method for treating and/or preventing a disease characterized by a cell of a subject being in a hypoxic state, comprising: administrating the pharmaceutical composition of the present invention to the subject. A known treatment method of a disease to be treated or prevented may be used concomitantly in the therapeutic and/or preventive method of the present invention.

[0047] In a further embodiment, the present invention relates to a use of the virus or viral vector of the present invention in manufacturing a medicament for treating and/or preventing a disease characterized by a cell being in a hypoxic state.

[0048] In another embodiment, the present invention relates to a fusion protein of an ODD and a protein essentially required for viral proliferation. The amino acid sequence of a fusion protein in which the protein essentially required for viral proliferation is HSV-1 ICP4 is shown in SEQ ID NO: 5. The fusion protein encompasses a protein which is composed of an amino acid sequence in which one or several, such as 9, 8, 7, 6, 5, 4, 3 or 2, of amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and which has the function of the fusion protein, namely the function of the fusion protein of an ODD and a protein essentially required for viral proliferation. The fusion protein of the present invention can be produced according to a known method such as genetic recombination. The fusion protein of the present invention can be used to label a hypoxic region in cells in vivo, by introducing into the cells in advance.

[0049] In still another embodiment, the present invention relates to a polynucleotide that encodes the above-mentioned fusion protein of an ODD and a protein essentially required for viral proliferation. The nucleic acid sequence that encodes a fusion protein in which the protein essentially required for viral proliferation is HSV-1 ICP4 is shown in SEQ ID NO: 6.

[0050] Although the following provides a specific and detailed explanation of the present invention by indicating the following examples thereof, the examples should not be understood to limit the present invention.

Example 1

Production of Virus

[0051] A polypeptide composed of 57 proteins containing the 564th proline residue, which is the ubiquitin-proteasome recognition site within the ODD (oxygen-dependent degradation domain) of HIF1.alpha., which is an amino acid sequence serving as a marker of protein degradation at normal oxygen atmospheric pressure, was added to the amino terminal of ICP4, which is a transcription factor essentially required for the initiation of HSV-1 replication. Moreover, a nuclear localization signal composed of 23 amino acids was added to the amino terminal thereof.

[0052] ICP4 gene was PCR-amplified using a 4085-bp blunt end SalI-MseI fragment (provided by Dr. Hayward of John Hopkins School of Medicine) derived from pGH108 (J. Virol., 56, 558-570, 1985) containing the coding region thereof, from the initiation codon to a PvuII site, and a 261-bp DNA encoding a Kozak sequence (aattcccagcttgac), a sequence of 23 amino acids serving as a nuclear localization signal, and 57 ODD sequences, was coupled to the 5' terminal thereof. A PvuII-MseI fragment of ICP4 was coupled to the 3' side thereof to construct an ODD-fused ICP4 gene (4221 bp). Moreover, an approximately 9-kb CMV-NLS-ODD-ICP4-IRES-LacZ-polyA fragment was constructed in which a CMV promoter (588 bp) was coupled to the upstream side thereof, while a LacZ-polyA sequence (3.3 kb) derived from E. coli was coupled to the downstream side thereof through IRES (585 bp). Moreover, a CMV-NLS-ODD-ICP4-IRES-EGFP-polyA fragment was also constructed in which LacZ-polyA was substituted to EGFP-polyA.

[0053] First, the CMV-NLS-ODD-ICP4-IRES-EGFP-polyA fragment was co-transfected with a drug resistance gene expression vector, pSV2neo, into a sub-confluent single-layer culture of human gastric cancer cell line AZP7a (provided by Dr. Nishimori of the Sapporo Medical University) in a 6-well tissue culture plate (2.5.times.10.sup.5/well) using Lipofectamine.RTM. (Gibco/BRL) in accordance with the manufacturer's protocol. G418-resistant clones of the gastric cancer cells that constitutively expressed EGFP were then selected using a method well known among persons with ordinary skill in the art. Whether or not expression of ICP4 protein changes depending on oxygen partial pressure was then examined using a fluorescence microscope under conditions of normal oxygen partial pressure (O.sub.2: 20%) and low oxygen partial pressure (O.sub.2: 1%) using the gastric cancer cell clones. Both EGFP and ICP4 were confirmed to be expressed within the same cells only under hypoxic conditions (FIG. 1). Although EGFP was expressed under both conditions of normal oxygen partial pressure (O.sub.2: 20%) and low oxygen partial pressure (O.sub.2: 1%), HIF1.alpha. and ICP4 were expressed only in cells cultured under conditions of low oxygen partial pressure (O.sub.2: 1%). Expression of ODD-ICP4 fusion protein was confirmed to be controlled according to the oxygen partial pressure of the cells (FIG. 1). Moreover, the CMV-NLS-ODD-ICP4-IRES-EGFP-polyA fragment and HSV-1 variant d120 viral DNA deficient in ICP4 were co-transfected into AZP7a cells in accordance with the manufacturer's protocol under conditions of normal oxygen partial pressure (O.sub.2: 20%) and low oxygen partial pressure (O.sub.2: 1%), and the number of plaques was counted after 48 hours. Plaques were confirmed to only have been formed under conditions of low oxygen partial pressure (O.sub.2: 1%) (FIG. 2). Plaque formation, which indicates viral proliferation, was observed to a much greater degree in the case of culturing at low oxygen partial pressure (O.sub.2: 1%). Not only the expression of ODD-ICP4 fusion protein (FIG. 1 and Western blot), but also the function thereof were confirmed to be controlled according to the oxygen partial pressure of the cells.

[0054] The CMV-NLS-ODD-ICP4-IRES-LacZ-polyA fragment was inserted into an StuI site of pKpX2 (J. Virol., 62, 196-205, 1998) (provided by Dr. Weller of Connecticut University), which contains a 2.3 kb sequence of the 5' side of a gene (UL39) that encodes RR, which is an enzyme essentially required for replication of viral DNA, followed by linearization by digesting with XhoI (in which the XbaI site further towards the 5' side of the RR sequence on the 5' side of pKpX2 and the HindIII site further towards the 3' side of the RR sequence on the 3' were substituted with XhoI) to construct an 11.3-kb UL39-CMV-NLS-ODD-ICP4-IRES-LacZ-polyA-UL39 homologous recombination vector from which pUC 19 sequence is removed. This was then co-transfected with HSV-1 variant d120 viral DNA deficient in ICP4 into a sub-confluent, single-layer culture of VeroE5 cells constitutively expressing ICP4 in a 6-well tissue culture plate (2.5.times.10.sup.5/well) using Lipofectamine.RTM. (Gibco/BRL) in accordance with the manufacturer's protocol. 1 ml of 20% DMEM culture medium was added 3 hours after transfection followed by culturing in the above-mentioned culture medium (10% FBS/DMEM) containing 0.5 mg/ml of 4-hydroxymethyl benzoic acid (HMBA) until 96 hours after transfection. Following confirmation of plaque formation, the cells were cultured for 24 hours in 10% FBS/DMEM not containing HMBA. The cells were then suspended in cold virus buffer (20 mM Tris-HCl containing 150 mM NaCl, pH: 7.5) at 500 .mu.l/well and placed in frozen storage.

[0055] Freezing and thawing treatment combined with ultrasonic treatment (3 times for 30 seconds each) were carried out three times to dissolve the above-mentioned suspension. The dissolved suspension was then stepwise-diluted and infected into sub-confluent, single-layer cultured VeroE5 cells in a 96-well tissue culture plate. Following infection, the cells were cultured for 96 hours in 1% FBS/DMEM containing 100 .mu.l of 11.3 .mu.g/ml human IgG (Jackson ImmunoResearch Laboratories) per well. VeroE5 single-layer cultured cells from those wells in which plaque formation was able to be confirmed were suspended in 100 .mu.l of the above-mentioned culture medium, and 6 .mu.l of the resulting suspension was used to measure .beta.-galactosidase enzyme activity using 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal) as the substrate and using a .beta. galactosidase enzyme assay system (Promega). Suspensions of VeroE5 cells from wells positive for .beta.-galactosidase enzyme activity were centrifuged for 5 minutes at 5000 rpm, and the resulting pellets were suspended in cold virus buffer at 100 .mu.l/well. Similar measurement of limited dilution infection/.beta.-galactosidase enzyme activity using a 96-well tissue culture plate was repeated two more times using VeroE5 cells to purify the recombinant virus d12.ODD.DELTA.RR as a single plaque (FIG. 3). The recombinant virus d12.ODD.DELTA.RR is maintained and managed by the inventors of the present invention.

[0056] Vero cells were infected in 10 to 20 150 cm.sup.2 tissue culture flasks (T-150, Iwaki Glass), and after culturing for 48 hours in VP-SMF serum-free medium (Invitrogen), separated cells were centrifuged for 45 minutes at 42000 g and recovered in the sediment fraction to prepare the virus. The cells were then suspended in 10 ml of cold virus buffer (20 mM Tris-HCl containing 150 mM NaCl, pH: 7.5). Freezing and thawing treatment combined with ultrasonic treatment (3 times for 30 seconds each) were carried out three times to dissolve the above-mentioned cells, followed by centrifuging for 5 minutes at 4.degree. C. and 500.times.g, and further centrifuging the supernatant for 45 minutes at 4.degree. C. and 26100.times.g. The resulting pellet was suspended in cold virus buffer, a plaque assay was carried out using Vero cells under conditions of normal oxygen partial pressure (O.sub.2: 20%) and low oxygen partial pressure (O.sub.2: 1%), and proliferating activity of the purified d12.ODD.DELTA.RR, sensitivity of its cell-injuring action to oxygen partial pressure, and its titer were determined. d12.ODD.DELTA.RR proliferated more actively and demonstrated cell-injuring activity under conditions of low oxygen partial pressure (O.sub.2: 1%) (FIG. 4).

[0057] Immunoblot Analysis of ICP4 Expression

[0058] AZP7a cells were respectively infected with d12.ODD.DELTA.RR to a multiplicity of infection (MOI) of 0.1 or only virus buffer under conditions of normal oxygen partial pressure (O.sub.2: 20%) and low oxygen partial pressure (O.sub.2: 1%), followed by recovery of the cells after culturing for 48 hours. An equal amount of protein was applied to SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). After blocking the membrane for 2 hours at room temperature using 5% skim milk (Difco Laboratories), the protein was incubated overnight at 4.degree. C. with anti-ICP4 antibody (Goodwin Institute for Cancer Research, dilution factor: 1:500). A larger amount of ICP4 protein was expressed under conditions of low oxygen partial pressure (O.sub.2: 1%) (FIG. 5). Expression of ICP4 was not observed with virus buffer only even after addition of AZP7a.

[0059] Virus Replication Analysis Indicating Ganciclovir Sensitivity of d12.ODD.DELTA.RR

[0060] Vero cell line was cultured in a 24-well culture plate at 5.times.10.sup.4 cells/well, and after infecting with d12.ODD.DELTA.RR virus at an MOI of 0.01, various concentrations (0 to 1 .mu.g/ml) of ganciclovir (Wako Pure Chemical Industries) in 1% FBS/DMEM were added followed by culturing for 26 hours. After fixing the cells with 10% formalin PBS, the resulting plaques were stained with X-Gal and counted. Proliferation of d12.ODD.DELTA.RR was inhibited concentration-dependently by addition of ganciclovir under both conditions of normal oxygen partial pressure (O.sub.2: 20%) and low oxygen partial pressure (O.sub.2: 1%). The results are shown in FIG. 6.

[0061] (In Vivo Treatment and Histological Analysis)

[0062] AZP7a cells were injected into the peritoneal cavities of 6-week-old, female severe combined immunodeficiency (SCID) mice (Nippon Clea), and MSTO human malignant mesothelioma cells (ATCC, CRL-2081), primary-cultured human leiomyoma cells and MCF7 human breast cancer cells (ATCC, HTB-22) were injected subcutaneously into the trunk to establish tumors in the mice. The MSTO cells were transfected with luciferase gene pGL4.13 (Promega), and clones demonstrating the highest chemiluminescence intensity and growth rate were selected. Among the cloned cells, tumor masses of the mesothelioma measuring 4 to 5 mm square were subcutaneously transplanted from those that had become established beneath the skin on the backs of the SCID mice to backs of 6-week-old, female SCID mice. The tumors grew to about 6 to 7 mm in diameter (50 to 70 mm.sup.3) at 30 days after being transplanted to the SCID mice. 50 .mu.l (per 100 mm.sup.3 of tumor volume) of virus suspension containing 1.times.10.sup.7 pfu of d12.ODD.DELTA.RR, or an equal volume of virus buffer, were injected intraperitoneally or intratumorally using a 30 gauge needle. The procedure was then repeated in exactly the same manner. Tumor diameter was measured at prescribed times after injection, and tumor volume was calculated using the following formula:

(tumor volume)=0.53.times.(length).times.(width).sup.2

In the case of MSTO, luciferin (Sigma Chemicals) was injected intraperitoneally, and in use of a high-sensitivity CCD camera the intensity of chemiluminescence from tumor cells beneath the skin of the back was measured employing a real-time in vivo imaging system (Berthold). d12.ODD.DELTA.RR demonstrated remarkable antitumor effects against each of the subcutaneously transplanted human malignant mesothelioma, leiomyoma and breast cancer cells by direct injection. The immunohistochemical results for human gastric cancer cells are shown in FIG. 7, while the treatment results for human malignant mesothelioma and leiomyoma are shown in FIGS. 8 and 9. Antitumor effects in a treatment group injected with d12.ODD.DELTA.RR a total of seven times on the prescribed days indicated with arrows were clearly evident in comparison with a control group injected with virus buffer only (FIG. 8). In contrast to residual tumor cells not being observed in tumors treated with d12.ODD.DELTA.RR, residual tumor cells were observed in tumors treated with d12.CALP.DELTA.RR (FIG. 9).

[0063] Mice in which tumors were present were sacrificed on prescribed numbers of days following completion of administration of d12.ODD.DELTA.RR at 1.times.10.sup.7 pfu/100 mm.sup.3 of tumor volume for the purpose of histological research. The subcutaneous tumors were excised and fixed overnight at 4.degree. C. in PBS containing 1 mM MgCl.sub.2 using 2% paraformaldehyde and 0.5% glutaraldehyde. Continuing, the tumors were immersed for 3 hours at 37.degree. C. in a substrate solution containing X-gal (1 mg/ml), 5 mM K.sub.3Fe(CN.sub.6), 5 mM K.sub.4Fe(CN.sub.6) and 1 mM MgCl.sub.2 in PBS, followed by washing with PBS containing 3% DMSO. Immunohistochemistry consisted of fixing the specimens in Bouin's solution (15% (v/v) saturated picric acid solution, 1.65% (v/v) formalin and 1% (v/v) acetic acid/PBS) followed by embedding in paraffin. A section having a thickness of 4 .mu.m was placed on a microslide coated with poly-L-lysine followed by treatment in xylene and stepwise dehydration with alcohol, and in order to block intrinsic peroxidase, the section was immersed in a solution of 70% methanol and H.sub.2O.sub.2. Subsequently, antigen was recovered in 10 mM citrate buffer (pH 7.0) using an autoclave for 10 minutes at 121.degree. C. The section was then incubated for 1 hour at room temperature using 1% (v/v) goat serum/PBS, followed by washing with PBS and incubating overnight at 4.degree. C. in 2% (w/v) BSA/PBS using the previously described anti-ICP4 antibody or anti-envelope antibody (Quartett). The section was then washed five times with 0.005% (v/v) Tween20/PBS followed by incubating for 1 hour at room temperature in 2% (w/v) BSA/PBS using biotinated goat anti-rabbit IgG (Tago Immunologicals), and further incubating for 30 minutes at room temperature using avidin-biotin-horseradish peroxidase conjugate (Vector Laboratories). After washing with 0.005% (v/v) Tween20/PBS, the final reaction product was visualized with diaminobentidine (Wako Chemicals), and the section was counter-stained with hematoxylin. A tissue specimen treated with goat serum was used as a control to observe non-specific staining. Detection of hypoxic regions of tissue was carried out using the Hypoxyprobe-1 Kit (Natural Pharmacia International) in accordance with the manufacturer's protocol. In an SCID mouse intraperitoneal transplant model of human gastric cancer cell line AZP7a, proliferation of intraperitoneally injected d12.ODD.DELTA.RR in coordination with hypoxic regions within tumors labeled with pimonidazole and expression of ICP4 protein were confirmed (FIG. 7).

[0064] After dividing cultured GIST cells into fractions positive and negative for CD133, which is a cell surface marker of cancer stem cells, using AutoMACS Pro (Miltenyi), the cells were cultured in Petri dishes and infected with purified d12.ODD.DELTA.RR at MOI of 0.01 to 0.0001 followed by analyzing virus replication, the results of which are shown in FIG. 10. d12.ODD.DELTA.RR proliferated more in CD133-positive cells and demonstrated potent cell-injuring action.

INDUSTRIAL APPLICABILITY

[0065] Since a virus or viral vector comprising a gene encoding a fusion protein of an ODD and a protein essentially required for viral proliferation, and a pharmaceutical composition comprising the same, are provided by the present invention, the present invention can be used in the development and production of therapeutic agents in the field of pharmaceuticals and the like, such as those for treatment of cancer under hypoxic conditions and resistant to radiotherapy and chemotherapy, and preferably cancer stem cells.

Sequence Listing Free Text

SEQ ID NO: 1: ODD

SEQ ID NO: 2: ODD

SEQ ID NO: 3: NLS-ODD

SEQ ID NO: 4: Kozak-NLS-ODD

[0066] SEQ ID NO: 5: ODD-ICP4 fusion protein SEQ ID NO: 6: Kozak-NLS-ODD-ICP4

Sequence CWU 1

1

6156PRTArtificial SequenceODD 1Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu Met Leu Ala1 5 10 15Pro Tyr Ile Asp Met Asp Asp Phe Gln Leu Arg Ser Phe Asp Gln Leu 20 25 30Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser Ala Ser Pro Gln 35 40 45Ser Thr Val Thr Val Phe Gln Gln 50 552171DNAArtificial SequenceODD 2aacccatttt ctactcagga cacagattta gacttggaga tgttagctcc ctatatccca 60atggatgatg acttccagtt acgttccttc gatcagttgt caccattaga aagcagttcc 120gcaagccctg aaagcgcaag tcctcaaagc acagttacag tattccagca g 171379PRTArtificial SequenceNLS-ODD 3Met Ala Pro Lys Lys Lys Arg Lys Arg Ser Tyr Gly Arg Lys Lys Arg1 5 10 15Arg Gln Arg Arg Arg Arg Ser Asn Pro Phe Ser Thr Gln Asp Thr Asp 20 25 30Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Asp Met Asp Asp Phe Gln 35 40 45Leu Arg Ser Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser 50 55 60Pro Glu Ser Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln65 70 754255DNAArtificial SequenceKozak-NLS-ODD 4aattcccagc ttgacatggc gcctaagaag aagaggaaga gatcatatgg tcgtaagaaa 60cgtcgccaac gtcgccgaag atctaaccca ttttctactc aggacacaga tttagacttg 120gagatgttag ctccctatat cccaatggat gatgacttcc agttacgttc cttcgatcag 180ttgtcaccat tagaaagcag ttccgcaagc cctgaaagcg caagtcctca aagcacagtt 240acagtattcc agcag 25551379PRTArtificial SequenceODD-ICP4 fusion protein 5Met Ala Pro Lys Lys Lys Arg Lys Arg Ser Tyr Gly Arg Lys Lys Arg1 5 10 15Arg Gln Arg Arg Arg Arg Ser Asn Pro Phe Ser Thr Gln Asp Thr Asp 20 25 30Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Asp Met Asp Asp Phe Gln 35 40 45Leu Arg Ser Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser 50 55 60Pro Glu Ser Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Val65 70 75 80Pro Met Ala Ser Glu Asn Lys Gln Arg Pro Gly Ser Pro Gly Pro Thr 85 90 95Asp Gly Pro Pro Pro Thr Pro Ser Pro Asp Arg Asp Glu Arg Gly Ala 100 105 110Leu Gly Trp Gly Ala Glu Thr Glu Glu Gly Gly Asp Asp Pro Asp His 115 120 125Asp Pro Asp His Pro His Asp Leu Asp Asp Ala Arg Arg Asp Gly Arg 130 135 140Ala Pro Ala Ala Gly Thr Asp Ala Gly Glu Asp Ala Gly Asp Ala Val145 150 155 160Ser Pro Arg Gln Leu Ala Leu Leu Ala Ser Met Val Glu Glu Ala Val 165 170 175Arg Thr Ile Pro Thr Pro Asp Pro Ala Ala Ser Pro Pro Arg Thr Pro 180 185 190Ala Phe Arg Ala Asp Asp Asp Asp Gly Asp Glu Tyr Asp Asp Ala Ala 195 200 205Asp Ala Ala Gly Asp Arg Ala Pro Ala Arg Gly Arg Glu Arg Glu Ala 210 215 220Pro Leu Arg Gly Ala Tyr Pro Asp Pro Thr Asp Arg Leu Ser Pro Arg225 230 235 240Pro Pro Ala Gln Pro Pro Arg Arg Arg Arg His Gly Arg Trp Arg Pro 245 250 255Ser Ala Ser Ser Thr Ser Ser Asp Ser Gly Ser Ser Ser Ser Ser Ser 260 265 270Ala Ser Ser Ser Ser Ser Ser Ser Asp Glu Asp Glu Asp Asp Asp Gly 275 280 285Asn Asp Ala Ala Asp His Ala Arg Glu Ala Arg Ala Val Gly Arg Gly 290 295 300Pro Ser Ser Ala Ala Pro Ala Ala Pro Gly Arg Thr Pro Pro Pro Pro305 310 315 320Gly Pro Pro Pro Leu Ser Glu Ala Ala Pro Lys Pro Arg Ala Ala Ala 325 330 335Arg Thr Pro Ala Ala Ser Ala Gly Arg Ile Glu Arg Arg Arg Ala Arg 340 345 350Ala Ala Val Ala Gly Arg Asp Ala Thr Gly Arg Phe Thr Ala Gly Gln 355 360 365Pro Arg Arg Val Glu Leu Asp Ala Asp Ala Thr Ser Gly Ala Phe Tyr 370 375 380Ala Arg Tyr Arg Asp Gly Tyr Val Ser Gly Glu Pro Trp Pro Gly Ala385 390 395 400Gly Pro Pro Pro Pro Gly Arg Val Leu Tyr Gly Gly Leu Gly Asp Ser 405 410 415Arg Pro Gly Leu Trp Gly Ala Pro Glu Ala Glu Glu Ala Arg Arg Arg 420 425 430Phe Glu Ala Ser Gly Ala Pro Ala Ala Val Trp Ala Pro Glu Leu Gly 435 440 445Asp Ala Ala Gln Gln Tyr Ala Leu Ile Thr Arg Leu Leu Tyr Thr Pro 450 455 460Asp Ala Glu Ala Met Gly Trp Leu Gln Asn Pro Arg Val Val Pro Gly465 470 475 480Asp Val Ala Leu Asp Gln Ala Cys Phe Arg Ile Ser Gly Ala Ala Arg 485 490 495Asn Ser Ser Ser Phe Ile Thr Gly Ser Val Ala Arg Ala Val Pro His 500 505 510Leu Gly Tyr Ala Met Ala Ala Gly Arg Phe Gly Trp Gly Leu Ala His 515 520 525Ala Ala Ala Ala Val Ala Met Ser Arg Arg Tyr Asp Arg Ala Gln Lys 530 535 540Gly Phe Leu Leu Thr Ser Leu Arg Arg Ala Tyr Ala Pro Leu Leu Ala545 550 555 560Arg Glu Asn Ala Ala Leu Thr Gly Ala Ala Gly Ser Pro Gly Ala Gly 565 570 575Ala Asp Asp Glu Gly Val Ala Ala Val Ala Ala Ala Ala Pro Gly Glu 580 585 590Arg Ala Val Pro Ala Gly Tyr Gly Ala Ala Gly Ile Leu Ala Ala Leu 595 600 605Gly Arg Leu Ser Ala Ala Pro Ala Ser Pro Ala Gly Gly Asp Asp Pro 610 615 620Asp Ala Ala Arg His Ala Asp Ala Asp Asp Asp Ala Gly Arg Arg Ala625 630 635 640Gln Ala Gly Arg Val Ala Val Glu Cys Leu Ala Ala Cys Arg Gly Ile 645 650 655Leu Glu Ala Leu Ala Glu Gly Phe Asp Gly Asp Leu Ala Ala Val Pro 660 665 670Gly Leu Ala Gly Ala Arg Pro Ala Ser Pro Pro Arg Pro Glu Gly Pro 675 680 685Ala Gly Pro Ala Ser Pro Pro Pro Pro His Ala Asp Ala Pro Arg Leu 690 695 700Arg Ala Trp Leu Arg Glu Leu Arg Phe Val Arg Asp Ala Leu Val Leu705 710 715 720Met Arg Leu Arg Gly Asp Leu Arg Val Ala Gly Gly Ser Glu Ala Ala 725 730 735Val Ala Ala Val Arg Ala Val Ser Leu Val Ala Gly Ala Leu Gly Pro 740 745 750Ala Leu Pro Arg Asp Pro Arg Leu Pro Ser Ser Ala Ala Ala Ala Ala 755 760 765Ala Asp Leu Leu Phe Asp Asn Gln Ser Leu Arg Pro Leu Leu Ala Ala 770 775 780Ala Ala Ser Ala Pro Asp Ala Ala Asp Ala Leu Ala Ala Ala Ala Ala785 790 795 800Ser Ala Ala Pro Arg Glu Gly Arg Lys Arg Lys Ser Pro Gly Pro Ala 805 810 815Arg Pro Pro Gly Gly Gly Gly Pro Arg Pro Pro Lys Thr Lys Lys Ser 820 825 830Gly Ala Asp Ala Pro Gly Ser Asp Ala Arg Ala Pro Leu Pro Ala Pro 835 840 845Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro Ala Pro Ala Gln Pro 850 855 860Ala Ala Pro Arg Ala Ala Ala Ala Gln Ala Arg Pro Arg Pro Val Ala865 870 875 880Val Ser Arg Arg Pro Ala Glu Gly Pro Asp Pro Leu Gly Gly Trp Arg 885 890 895Arg Gln Pro Pro Gly Pro Ser His Thr Ala Ala Pro Ala Ala Ala Ala 900 905 910Leu Glu Ala Tyr Cys Ser Pro Arg Ala Val Ala Glu Leu Thr Asp His 915 920 925Pro Leu Phe Pro Val Pro Trp Arg Pro Ala Leu Met Phe Asp Pro Arg 930 935 940Ala Leu Ala Ser Ile Ala Ala Arg Cys Ala Gly Pro Ala Pro Ala Ala945 950 955 960Gln Ala Ala Cys Gly Gly Gly Asp Asp Asp Asp Asn Pro His Pro His 965 970 975Gly Ala Ala Gly Gly Arg Leu Phe Gly Pro Leu Arg Ala Ser Gly Pro 980 985 990Leu Arg Arg Met Ala Ala Trp Met Arg Gln Ile Pro Asp Pro Glu Asp 995 1000 1005Val Arg Val Val Val Leu Tyr Ser Pro Leu Pro Gly Glu Asp Leu 1010 1015 1020Ala Gly Gly Gly Ala Ser Gly Gly Pro Pro Glu Trp Ser Ala Glu 1025 1030 1035Arg Gly Gly Leu Ser Cys Leu Leu Ala Ala Leu Ala Asn Arg Leu 1040 1045 1050Cys Gly Pro Asp Thr Ala Ala Trp Ala Gly Asn Trp Thr Gly Ala 1055 1060 1065Pro Asp Val Ser Ala Leu Gly Ala Gln Gly Val Leu Leu Leu Ser 1070 1075 1080Thr Arg Asp Leu Ala Phe Ala Gly Ala Val Glu Phe Leu Gly Leu 1085 1090 1095Leu Ala Ser Ala Gly Asp Arg Arg Leu Ile Val Val Asn Thr Val 1100 1105 1110Arg Ala Cys Asp Trp Pro Ala Asp Gly Pro Ala Val Ser Arg Gln 1115 1120 1125His Ala Tyr Leu Ala Cys Glu Leu Leu Pro Ala Val Gln Cys Ala 1130 1135 1140Val Arg Trp Pro Ala Ala Arg Asp Leu Arg Arg Thr Val Leu Ala 1145 1150 1155Ser Gly Arg Val Phe Gly Pro Gly Val Phe Ala Arg Val Glu Ala 1160 1165 1170Ala His Ala Arg Leu Tyr Pro Asp Ala Pro Pro Leu Arg Leu Cys 1175 1180 1185Arg Gly Gly Asn Val Arg Tyr Arg Val Arg Thr Arg Phe Gly Pro 1190 1195 1200Asp Thr Pro Val Pro Met Ser Pro Arg Glu Tyr Arg Arg Ala Val 1205 1210 1215Leu Pro Ala Leu Asp Gly Arg Ala Ala Ala Ser Gly Thr Thr Asp 1220 1225 1230Ala Met Ala Pro Gly Ala Pro Asp Phe Cys Glu Glu Glu Ala His 1235 1240 1245Ser His Ala Ala Cys Ala Arg Trp Gly Leu Gly Ala Pro Leu Arg 1250 1255 1260Pro Val Tyr Val Ala Leu Gly Arg Glu Ala Val Arg Ala Gly Pro 1265 1270 1275Ala Arg Trp Arg Gly Pro Arg Arg Asp Phe Cys Ala Arg Ala Leu 1280 1285 1290Leu Glu Pro Asp Asp Asp Ala Pro Pro Leu Val Leu Arg Gly Asp 1295 1300 1305Asp Asp Gly Pro Gly Ala Leu Pro Pro Ala Pro Pro Gly Ile Arg 1310 1315 1320Trp Ala Ser Ala Thr Gly Arg Ser Gly Thr Val Leu Ala Ala Ala 1325 1330 1335Gly Ala Val Glu Val Leu Gly Ala Glu Ala Gly Leu Ala Thr Pro 1340 1345 1350Pro Arg Arg Glu Val Val Asp Trp Glu Gly Ala Trp Asp Glu Asp 1355 1360 1365Asp Gly Gly Ala Phe Glu Gly Asp Gly Val Leu 1370 137564220DNAArtificial SequenceKozak-NLS-ODD-ICP4 6aattcccagc ttgacatggc gcctaagaag aagaggaaga gatcatatgg tcgtaagaaa 60cgtcgccaac gtcgccgaag atctaaccca ttttctactc aggacacaga tttagacttg 120gagatgttag ctccctatat cccaatggat gatgacttcc agttacgttc cttcgatcag 180ttgtcaccat tagaaagcag ttccgcaagc cctgaaagcg caagtcctca aagcacagtt 240acagtattcc agcaggtacc aatggcgtcg gagaacaagc agcgccccgg ctccccgggc 300cccaccgacg ggccgccgcc caccccgagc ccagaccgcg acgagcgggg ggccctcggg 360tggggcgcgg agacggagga gggcggggac gaccccgacc acgaccccga ccacccccac 420gacctcgacg acgcccggcg ggacgggagg gcccccgcgg cgggcaccga cgccggcgag 480gacgccgggg acgccgtctc gccgcgacag ctggctctgc tggcctccat ggtagaggag 540gccgtccgga cgatcccgac gcccgacccc gcggcctcgc cgccccggac ccccgccttt 600cgagccgacg acgatgacgg ggacgagtac gacgacgcag ccgacgccgc cggcgaccgg 660gccccggccc ggggccgcga acgggaggcc ccgctacgcg gcgcgtatcc ggaccccacg 720gaccgcctgt cgccgcgccc gccggcccag ccgccgcgga gacgtcgtca cggccggtgg 780cggccatcgg cgtcatcgac ctcgtcggac tccgggtcct cgtcctcgtc gtccgcatcc 840tcttcgtcct cgtcgtccga cgaggacgag gacgacgacg gcaacgacgc ggccgaccac 900gcacgcgagg cgcgggccgt cgggcggggt ccgtcgagcg cggcgccggc agcccccggg 960cggacgccgc ccccgcccgg gccacccccc ctctccgagg ccgcgcccaa gccccgggcg 1020gcggcgagga cccccgcggc ctccgcgggc cgcatcgagc gccgccgggc ccgcgcggcg 1080gtggccggcc gcgacgccac gggccgcttc acggccgggc agccccggcg ggtcgagctg 1140gacgccgacg cgacctccgg cgccttctac gcgcgctatc gcgacgggta cgtcagcggg 1200gagccgtggc ccggcgccgg gcccccgccc ccggggcggg tgctgtacgg cggcctgggc 1260gacagccgcc cgggcctctg gggggcgccc gaggcggagg aggcgcgacg ccggttcgag 1320gcctcgggcg ccccggcggc cgtgtgggcg cccgagctgg gcgacgccgc gcagcagtac 1380gccctgatca cgcggctgct gtacaccccg gacgcggagg ccatggggtg gctccagaac 1440ccgcgcgtgg tccccgggga cgtggcgctg gaccaggcct gcttccggat ctcgggcgcc 1500gcgcgcaaca gcagctcctt catcaccggc agcgtggcgc gggccgtgcc ccacctgggc 1560tacgccatgg cggccggccg cttcggctgg ggcctggcgc acgcggcggc cgccgtggcc 1620atgagccgcc gatacgaccg cgcgcagaag ggcttcctgc tgaccagcct gcgccgcgcc 1680tacgcgcccc tgttggcgcg cgagaacgcg gcgctgacgg gggccgcggg gagccccggc 1740gccggcgcag atgacgaggg ggtcgccgcc gtcgccgccg ccgcaccggg cgagcgcgcg 1800gtgcccgccg ggtacggcgc cgcggggatc ctcgccgccc tggggcggct gtccgccgcg 1860cccgcctccc ccgcgggggg cgacgacccc gacgccgccc gccacgccga cgccgacgac 1920gacgccgggc gccgcgccca ggccggccgc gtggccgtcg agtgcctggc cgcctgccgc 1980gggatcctgg aggcgctggc cgagggcttc gacggcgacc tggcggccgt cccggggctg 2040gccggggccc ggcccgccag ccccccgcgg ccggagggac ccgcgggccc cgcttccccg 2100ccgccgccgc acgccgacgc gccccgcctg cgcgcgtggc tgcgcgagct gcggttcgtg 2160cgcgacgcgc tggtgctcat gcgcctgcgc ggggacctgc gcgtggccgg cggcagcgag 2220gccgccgtgg ccgccgtgcg cgccgtgagc ctggtcgccg gggccctggg ccccgcgctg 2280ccgcgggacc cgcgcctgcc gagctccgcg gccgccgccg ccgcggacct gctgtttgac 2340aaccagagcc tgcgccccct gctggcggcg gcggccagcg caccggacgc cgccgacgcg 2400ctggcggccg ccgccgcctc cgccgcgccg cgggaggggc gcaagcgcaa gagtcccggc 2460ccggcccggc cgcccggagg cggcggcccg cgacccccga agacgaagaa gagcggcgcg 2520gacgcccccg gctcggacgc ccgcgccccc ctccccgcgc ccgcgccccc ctccacgccc 2580ccggggcccg agcccgcccc cgcccagccc gcggcgcccc gggccgccgc ggcgcaggcc 2640cgcccgcgcc ccgtggccgt gtcgcgccgg cccgccgagg gccccgaccc cctgggcggc 2700tggcggcggc agcccccggg gcccagccac acggcggcgc ccgcggccgc cgccctggag 2760gcctactgct ccccgcgcgc cgtggccgag ctcacggacc acccgctgtt ccccgtcccc 2820tggcgaccgg ccctcatgtt tgacccgcgg gccctggcct cgatcgccgc gcggtgcgcc 2880gggcccgccc ccgccgccca ggccgcgtgc ggcggcggcg acgacgacga taacccccac 2940ccccacgggg ccgccggggg ccgcctcttt ggccccctgc gcgcctcggg cccgctgcgc 3000cgcatggcgg cctggatgcg ccagatcccc gaccccgagg acgtgcgcgt ggtggtgctg 3060tactcgccgc tgccgggcga ggacctggcc ggcggcgggg cctcgggggg gccgccggag 3120tggtccgccg agcgcggcgg gctgtcctgc ctgctggcgg ccctggccaa ccggctgtgc 3180gggccggaca cggccgcctg ggcgggcaat tggaccggcg cccccgacgt gtcggcgctg 3240ggcgcacagg gcgtgctgct gctgtccacg cgggacctgg ccttcgccgg ggccgtggag 3300tttctggggc tgctcgccag cgccggcgac cggcggctca tcgtggtcaa caccgtgcgc 3360gcctgcgact ggcccgccga cgggcccgcg gtgtcgcggc agcacgccta cctggcgtgc 3420gagctgctgc ccgccgtgca gtgcgccgtg cgctggccgg cggcgcggga cctgcgccgc 3480acggtgctgg cctcgggccg cgtgttcggc ccgggggtct tcgcgcgcgt ggaggccgcg 3540cacgcgcgcc tgtaccccga cgcgccgccg ctgcgcctgt gccgcggcgg caacgtgcgc 3600taccgcgtgc gcacgcgctt cggcccggac acgccggtgc ccatgtcccc gcgcgagtac 3660cgccgggccg tgctgccggc gctggacggc cgggcggcgg cctcggggac caccgacgcc 3720atggcgcccg gcgcgccgga cttctgcgag gaggaggccc actcgcacgc cgcctgcgcg 3780cgctggggcc tgggcgcgcc gctgcggccc gtgtacgtgg cgctggggcg cgaggcggtg 3840cgcgccggcc cggcccggtg gcgcgggccg cggagggact tttgcgcccg cgccctgctg 3900gagcccgacg acgacgcccc cccgctggtg ctgcgcggcg acgacgacgg cccgggggcc 3960ctgccgccgg cgccgcccgg gattcgctgg gcctcggcca cgggccgcag cggcaccgtg 4020ctggcggcgg cgggggccgt ggaggtgctg ggggcggagg cgggcttggc cacgcccccg 4080cggcgggaag ttgtggactg ggaaggcgcc tgggacgaag acgacggcgg cgcgttcgag 4140ggggacgggg tgctgtaacg ggccgggacg gggcggggcg cttgtgagac ccgaagacgc 4200aataaacggc aacaacctga 4220

Claims

1-16. (canceled)

17. A method for treating or preventing a disease comprising administering to a subject a virus or viral vector, which is obtainable by a method for producing a virus or viral vector comprising the steps of: (a) infecting a cell with a virus or viral vector, comprising a gene encoding a fusion protein of an oxygen-dependent degradation domain (ODD) and a protein essentially required for viral proliferation, (b) culturing the cell in a hypoxic state, and (c) recovering the virus that has proliferated, wherein the disease is characterized by a cell in the subject having replicating ability in a hypoxic state.